Compositions for establishing mixed chimerism and methods of manufacture thereof

ABSTRACT

The invention provides compositions for establishing mixed chimerism in a subject. The compositions include CD34+ cells that have been column-purified from an apheresis product and CD3+ cells from an apheresis product that have not been purified through a column. The invention also provides methods of making and using such compositions.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of, and priority to, U.S.Non-provisional Patent Application No. 15/946,099, filed Apr. 5, 2018,the contents of which are incorporated by reference.

FIELD OF THE INVENTION

The invention relates to compositions for establishing mixed chimerismin a subject and methods of manufacture and use thereof.

BACKGROUND

Over 30,000 people receive organ transplants annually in the UnitedStates. Although an organ transplant can save or transform therecipient's life, transplantation remains a risky procedure. A majorconcern is that the recipient's immune system will identify thetransplanted organ as foreign and destroy it. For example, the rate ofrejection for kidney transplants, the most common type of solid organtransplant, is about 25%. Consequently, most transplant recipients musttake immunosuppressive drugs for the rest of their lives.Immunosuppressive therapy, however, carries its own set of risks,including increased risk of infection, cancer, hypertension, and liverdamage.

To date, the only way to maintain long-term graft tolerance in humanswithout immunosuppression is to reconstruct the recipient's immunesystem to include a mixture of donor-derived and recipient-derivedhematopoietic cells. Mixed chimerism is established by providinghematopoietic cellular compositions that include CD34⁺ and CD3⁺ cells(See Strober et al., U.S. Pat. Nos. 9,504,717 and 9,561,253). Themanufacture of such hematopoietic cellular compositions starts byobtaining an apheresis product from an organ donor. The apheresisproduct includes CD34⁺ and CD3⁺ cells. After certain initial work-upsteps, the apheresis product is purified to enrich for the CD34⁺ cellsin the apheresis product. To enrich for the CD34⁺ cells, the entireapheresis product is flowed through an immunomagnetic-bead column, e.g.,an Isolex column. The CD34⁺ cells are retained by theimmunomagnetic-bead column. The column flow through of the apheresisproduct (column effluent) is collected and includes the CD3⁺ cells,which are not retained by the immunomagnetic-bead column. The enrichedCD34⁺ cells are then eluted from the immunomagnetic-bead column. Adefined amount of the column effluent having the CD3⁺ cells is thenadded to the enriched CD34⁺ cells until a desired concentration of CD3⁺cells is achieved.

SUMMARY

The invention recognizes that there is a need for a more commerciallyamenable high throughput manufacturing process for making hematopoieticcellular compositions. The invention provides new methods formanufacturing hematopoietic cellular compositions and new hematopoieticcellular compositions for establishing mixed chimerism. Particularly,the invention provides an approach in which an entire apheresis productis not flowed through a CD34⁺ cell enriching column. Rather than flowingthe entire apheresis product through a CD34⁺ cell enriching column, theapheresis product, which includes CD34⁺ and CD3⁺ cells, is divided intoa first portion and a second portion. The first portion of the apheresisproduct is purified, e.g., by flow through the CD34⁺ cell enrichingcolumn, to obtain an enriched amount of CD34⁺ cells. The second portionof the apheresis product is not flowed through the CD34⁺ cell enrichingcolumn. To complete the manufacture of the hematopoietic cellularcompositions of the invention, at least some of the second portion ofthe apheresis product, comprising CD34⁺ and CD3⁺ cells, is introduced tothe enriched amount of CD34⁺ cells. In this approach, the CD3⁺ cellsthat make-up the hematopoietic cellular compositions of the inventionare not flowed through a CD34⁺ cell enriching column and in fact neverinteract with the CD34⁺ cell enriching column. Importantly, the columneffluent does not need to be retained for the hematopoietic cellularcompositions of the invention. By obviating the need to retain thecolumn effluent, the methods can more easily be modified for large-scaleproduct preparation. In that manner, the invention provides commercialmanufacturing methods that are robust, efficient, scalable, andreproducible, while also providing hematopoietic cellular compositionsin which the CD3⁺ cells that make-up the hematopoietic cellularcompositions of the invention are not flowed through a CD34⁺ cellenriching column.

In certain aspects, the invention provides cellular products forestablishing mixed chimerism in a solid organ transplant recipient. Thecellular products include column-purified CD34⁺ cells derived from anapheresis product and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column. In other aspects, theinvention provides cellular products including column purified CD34⁺cells derived from an apheresis product, and CD3⁺ cells derived from theapheresis product that have not passed through a purification column. Inother aspects, the invention provides cellular products includingcomponent A and component B, in which component A includes CD34⁺ cellsthat have been purified from a first portion of a non-column purifiedapheresis product, and in which component B includes a second portion ofthe non-column purified apheresis product including CD3⁺. Unlike priorhematopoietic cellular compositions that included purified CD34⁺ cellscombined with column effluent including CD3⁺ cells, the hematopoieticcellular compositions of the invention do not include column effluent.

In another aspect, the invention provides methods for manufacturing acellular product for establishing mixed chimerism in a solid organtransplant recipient. The methods include receiving an apheresis productcontaining CD34⁺ and CD3⁺ cells; dividing the apheresis product into afirst portion and a second portion; purifying the first portion toobtain an enriched amount of CD34⁺ cells; and introducing at least someof the second portion containing CD3⁺ cells to the enriched amount ofCD34⁺ cells.

The compositions of the invention can include various concentrations foreach of the CD34⁺ cells and CD3⁺ cells, and different concentrations arediscussed herein. The amount may be specified as a number of cellsrelative to the body mass of the recipient. For example, the cellularproduct may contain at least 1×10⁵, 2×10⁵, 5×10⁵, 1×10⁶, 2×10⁶, or 4×10⁶CD34⁺ cells/kg recipient weight. The cellular product may contain atleast 1×10⁴, 2×10⁴, 5×10⁴, 1×10⁵, 2×10⁵, 5×10⁵, 1×10⁶, 2×10⁶, 5×10⁶,1×10⁷, 2×10⁷, 5×10⁷, or 1×10⁸ CD3⁺ cells/kg recipient weight.

The CD34⁺ cells and CD3⁺ cells may be provided in separate containers.The CD34⁺ cells and CD3⁺ cells may be provided as a mixture in the samecontainer. The cellular product may be cryopreserved. The cellularproduct may contain one or more cryoprotectants. The cryoprotectant maybe dextran having an average molecular weight of 40,000 Da or DMSO. Thecellular product may contain the cryoprotectant at a concentration ofabout 1%, 2%, 3%, 4%, 5%, 7.5%, or 10%.

The CD34⁺ cells, the CD3⁺ cells, or both may be derived from the solidorgan transplant donor. The donor and the recipient may be HLA-matched,or they may be HLA-mismatched. The donor and recipient may beHLA-matched at six, eight, ten, or twelve alleles among the HLA-A,HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR genes. The donor and recipientmay be HLA-mismatched at one, two, three, four, five, six, or morealleles among the HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR genes.

The CD34⁺ cells may be column-purified by any acceptable cellpurification method. For example, the CD34⁺ cells may be immunopurifiedusing immunomagnetic particles and a magnetic column.

The CD34⁺ cells and CD3⁺ cells may be obtained from a single apheresisproduct. The CD34⁺ cells and CD3⁺ cells may be obtained from multipleapheresis products, for example, two, three, four, five, six, or moreapheresis products. The CD34⁺ cells and CD3⁺ cells may be obtained froma cryopreserved apheresis product. The CD34⁺ cells may becolumn-purified prior to cryopreservation or after cryopreservation.

DETAILED DESCRIPTION

The primary hurdle in organ transplantation is getting the recipient totolerate the donor's tissue. If the recipient's immune system detectsthe donated organ as foreign, it attacks the tissue, leading to graftrejection. Consequently, most transplant recipients must take drugs thatsuppress the immune system. Immunosuppressive therapy, however, createsits own set of risks. For example, immunosuppressive drugs decrease thebody's ability to ward off infections. In addition, because they hinderthe immune system's ability to identify and destroy malignant tissue,immunosuppressive drugs increase the risk of developing cancer.

To avoid graft rejection, transplantation of solid organs may beaccompanied by transfer of donor-derived blood cell progenitors.Providing donor blood cells allows reconstitution of the recipient'simmune system to include cells that have been educated to recognize theorgan as non-foreign tissue. Consequently, the donated organ is notattacked, and the recipient tolerates the graft.

One strategy for reconstructing the recipient's immune system entailscomplete replacement of the recipient's hematopoietic system withexclusively donor-derived cells to achieve a state of full chimerism. Arisk associated with full chimerism, however, is that the completelydonor-derived immune system may identify the recipient's tissue asforeign and attack it, a condition called graft-versus-host disease(GVHD). See, e.g., Sach et al., Induction of Tolerance through MixedChimerism, Cold Spring Harb Perspect Med 2014; 4:a015529, doi:10.1101/cshperspect.a015529, the contents of which are incorporatedherein by reference. As a result, fully chimeric patients must remain onimmunosuppressive therapy indefinitely.

Another strategy is to repopulate the recipient's immune system with amixture of donor-derived cells and recipient-derived cells to attain astate called mixed chimerism. Compared to full chimerism, mixedchimerism is associated with lower rates of GVHD. In addition, mixedchimeric regimens require lower doses of immunosuppressive therapyinitially and allow complete discontinuation of immunosuppression afterthe stability of the recipient's mixed chimerism has been established.To date, induction of mixed chimerism is the only method of producinggraft tolerance in humans without maintaining immunosuppressive therapy.

The compositions of the invention are useful for establishing mixedchimerism in a solid organ transplant recipient. It is recognized in theart that providing donor-derived CD34⁺ cells and CD3⁺ cells inconjunction with solid organ transplants facilitates the maintenance ofmixed chimerism. The present invention provides improved compositionsfor establishing mixed chimerism in solid organ transplant recipientsand methods of making and using such compositions.

Cellular Products

All blood cells, including the cells of the immune system, are derivedfrom hematopoietic stem cells (HSCs). HSCs are multipotent cells thatcan differentiate into various specialized cells and also reproduce togenerate new HSCs. HSCs that differentiate form either lymphoidprogenitors or myeloid progenitors. Lymphoid progenitors give rise tolymphocytes and natural killer cells. Myeloid progenitors produce cellsof the myeloid and erythroid lineages, such as erythrocytes, platelets,basophils, neutrophils, eosinophils, monocytes, macrophages, andantigen-presenting cells, such as dendritic cells. In adults, mosthematopoietic development occurs in the bone marrow, although maturationand activation of some lymphoid cells occurs in the spleen, thymus, andlymph nodes.

The cellular products of the invention include two populations of cellsthat allow donor HSCs to develop into mature cells of the immune systemin the recipient's body. One population includes CD34⁺ cells. CD34 is acell surface marker that is expressed in HSCs and their immediatedescendants, multipotent progenitor cells, which have not committed toeither the myeloid or lymphoid lineage. Consequently, CD34 expression isa useful measure for identifying populations of cells that contain HSCs.The other population includes CD3⁺ cells. CD3 comprises a group ofpolypeptides that interact with the two polypeptide chains of the T cellreceptor to form the T cell receptor complex. The CD3 complex includes agamma chain, delta chain, and two epsilon chains. CD3 is expressed onthe surface of mature T cells and is thus useful as a marker for Tcells.

To promote establishment of mixed chimerism in the recipient, thecellular products include CD34⁺ cells and CD3⁺ cells in appropriatequantities. For example, an ample supply of CD34⁺ cells is necessary todevelop a stable population of donor-derived immune cells in therecipient. However, CD34⁺ cells are relatively scarce, making up onlyabout 0.1-0.2% of peripheral blood cells in normal, untreated patients.Therefore, the cellular products contain CD34⁺ cells that have beencolumn purified from an apheresis product to obtain a sufficient numberof such cells. In contrast, CD3⁺ cells are abundant, accounting for amajority of mononuclear cells in the peripheral blood. Thus, thepopulation of CD3⁺ cells in the cellular products is taken from aportion of the apheresis product that has not been subjected to a columnpurification step. Avoiding unnecessary manipulation of the CD3⁺population used in the cellular products has several advantages. First,it minimizes the opportunity for cells to become damaged and thereforepreserves cell viability. In addition, it allows the CD3⁺ fraction toretain circulating factors from the donor's blood that may facilitatehematopoiesis in the recipient. Finally, it simplifies the preparationof the cellular products.

The cellular products may contain CD34⁺ cells and CD3⁺ cells in definedamounts. A useful unit of cell quantity in a product is the number ofcells relative to the body mass of the recipient. For example andwithout limitation, the cellular product may contain at least 1×10⁴,2×10⁴, 5×10⁴, 1×10⁵, 2×10⁵, 5×10⁵, 1×10⁶, 2×10⁶, or 4×10⁶, 1×10⁷, 2×10⁷,4×10⁷, or 1×10⁸ CD34⁺ cells/kg recipient weight. For example and withoutlimitation, the cellular product may contain at least 1×10⁴, 2×10⁴,5×10⁴, 1×10⁵, 2×10⁵, 5×10⁵, 1×10⁶, 2×10⁶, 5×10⁶, 1×10⁷, 2×10⁷, 5×10⁷, or1×10⁸ CD3⁺ cells/kg recipient weight. Other concentrations areexemplified in each of Strober et al., U.S. Pat. No. 9,504,717 andStrober et al., U.S. Pat. No. 9,561,253, the content of each of which isincorporated by reference herein in its entirety.

The cellular product may contain CD34⁺ cells at a designated level ofpurity. For example, the cellular product may contain CD34⁺ cells thatare at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least99% pure. Other purities are exemplified in each of Strober et al., U.S.Pat. No. 9,504,717 and Strober et al., U.S. Pat. No. 9,561,253, thecontent of each of which is incorporated by reference herein in itsentirety.

The CD34⁺ cells and CD3⁺ cells may be derived from any donor. The CD34⁺cells and CD3⁺ cells may be from the same donor. The CD34⁺ cells andCD3⁺ cells may be from different donors. Preferably, the CD34⁺ cells andCD3⁺ cells are derived from the donor of the solid organ that is beingor has been transplanted into the recipient. The CD34⁺ cells and CD3⁺cells may be derived from a living donor. The CD34⁺ cells and CD3⁺ cellsmay be derived from a deceased donor.

The CD34⁺ cells and CD3⁺ cells may be provided as a mixture in one ormore containers. The CD34⁺ cells and CD3⁺ cells may be provided inseparate container. Any commercially available container approved tohold cellar products may be used.

The cellular product may be provided frozen. Consequently, the cellularproduct may contain a cryoprotectant. Any cryoprotectant known in theart may be used. For example and without limitation, the cryoprotectantmay be DMSO, dextran having an average molecular weight of 40 kDa,serum, e.g., bovine serum, albumin, e.g., human serum albumin, or cellculture medium. The cryoprotectant may be present at a definedconcentration. For example, the cellular product may contain about 1%DMSO, about 2% DMSO, about 5% DMSO, about 7.5% DMSO, about 10% DMSO,about 12.5% DMSO, about 15% DMSO, or about 20% DMSO. The cellularproduct may contain about 1% dextran, about 2% dextran, about 5%dextran, about 7.5% dextran, about 10% dextran, about 12.5% dextran,about 15% dextran, or about 20% dextran. Cyroprotection is discussed ineach of Strober et al., U.S. Pat. No. 9,504,717 and Strober et al., U.S.Pat. No. 9,561,253, the content of each of which is incorporated byreference herein in its entirety.

The cellular product may contain agents that enhance engraftment orfunctional mobilization of the hematopoietic cells in the recipient. Thecellular product may contain agents that prevent a negative reaction ofthe recipient to the hematopoietic cells. For example and withoutlimitation, the pharmaceutical composition may contain a cytokine,chemokine, growth factor, excipient, carrier, antibody or a fragmentthereof, small molecule, drug, agonist, antagonist, matrix protein, orcomplementary cell type.

The cellular product may contain a buffer. The cellular product may bebuffer to maintain physiologically compatible pH. For example, thecellular product may be buffered to a neutral pH, such as from about 6.0to about 8.0.

The cellular product can be supplied in the form of a pharmaceuticalcomposition, comprising an isotonic excipient prepared undersufficiently sterile conditions for human administration. Choice of thecellular excipient and any accompanying elements of the composition isadapted in accordance with the route and device used for administration.For general principles in medicinal formulation, see Cell Therapy: StemCell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G.Morstyn & W. Sheridan. eds., Cambridge University Press, 1996; andHematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law,Churchill Livingstone, 2000.

The donor cells may be HLA-matched or HLA-mismatched to the recipient.Human leukocyte antigens (HLAs), also called major histocompatibilitycomplex (MHC) antigens, are protein molecules expressed on the surfaceof cells that confer a unique antigenic identity to these cells. MHC/HLAantigens are target molecules that are recognized by T-cells and naturalkiller (NK) cells as being derived from the same source of hematopoieticstem cells as the immune effector cells (“self”) or as being derivedfrom another source of hematopoietic reconstituting cells (“non-self”).Two main classes of HLA antigens are recognized: HLA class I and HLAclass II. HLA class I antigens (A, B, and C in humans) render each cellrecognizable as “self,” whereas HLA class II antigens (DR, DP, and DQ inhumans) are involved in reactions between lymphocytes and antigenpresenting cells.

A key aspect of the HLA gene system is its polymorphism. Each geneexists in different alleles. Allelic gene products differ in one or moreamino acids in the alpha and/or beta domain(s). An individual has twoalleles of each gene, for a total of twelve alleles among the HLA-A,HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR genes. An HLA-matched donor mayhave a match with the recipient at six, eight, ten, or twelve allelesselected from any combination of the HLA-A, HLA-B, HLA-C, HLA-DP,HLA-DQ, and HLA-DR genes. The genes most important for HLA typing areHLA-A, HLA-B, and HLA-DR, so the donor and recipient may be matched atall six alleles of the HLA-A, HLA-B, and HLA-DR genes. An HLA-mismatcheddonor may have a mismatch at one, two, three, four, five, six, or morealleles among the HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR genes.HLA typing may be performed by any method known in the art. Examples ofHLA typing methods include serological cytotoxicity, flow cytometry, andDNA typing. Such methods are described in, for example, U.S. Pat. No.9,561,253, the contents of which are incorporated herein by reference.

The HLA genes are clustered in a super-locus present on chromosomeposition 6p21. Consequently, the set of alleles present on a singlechromosome, i.e., a haplotype, tends to be inherited as a group.Identifying a patient's haplotypes can help predict the probability offinding matching donors and assist in developing a search strategy.Haplotypes vary in how common they are among the general population andin their frequency within different racial and ethnic groups.

Numerous exemplary embodiments are now described below, both HLA matchedand HLA mismatched. The skilled artisan will recognize that the belowembodiments are exemplary and non-limiting, particularly, the belowembodiments do not limit any other part or exemplified cell amounts orcombinations in any other part of this application.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of 1×10⁶ CD3⁺cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cells beingHLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁸ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁹ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁸ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10 ⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁸ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁸ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁸ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-matched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 5×10⁵ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁸ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 1×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁸ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 2×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁸ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁵ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁶ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least2×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least5×10⁷ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

The cellular product may contain column-purified CD34⁺ cells derivedfrom an apheresis product in an amount of at least 4×10⁶ CD34⁺ cells/kgrecipient weight and CD3⁺ cells derived from the apheresis product thathave not passed through a purification column in an amount of at least1×10⁸ CD3⁺ cells/kg recipient weight, the CD34⁺ cells and CD3⁺ cellsbeing HLA-mismatched to the recipient.

Preparation of Cellular Products

The cellular products of the invention are prepared from one or moreapheresis products. Preferably, the one or more apheresis products aredivided into two portions: one portion for column-purification of CD34⁺cells, and one portion to serve as the source of CD3⁺ cells. Whenmultiple apheresis products are used, they may be combined and thendivided into portions. Alternatively, individual apheresis products canbe divided into portions, and the portions from individual apheresisproducts can be combined. Preferably, the apheresis products areobtained from the solid organ donor. Apheresis methods are known in theart and described in, for example, U.S. Pat. No. 9,561,253, the contentsof which are incorporated herein by reference.

As indicated above, CD34⁺ cells make up a low percentage of peripheralblood cells in normal subjects. However, the fraction of CD34⁺ cells inblood can be increased by administering to the subject a factor, such asgranulocyte colony stimulating factor (G-CSF), that mobilizes CD34⁺cells from bone marrow and other sources. Thus, prior to apheresis, thesubject may be given G-CSF to mobilize CD34⁺ cells. Regimens foradministering G-CSF to a subject prior to apheresis, including thedosage, frequency, and timing of administration, are known in the artand described in, for example, U.S. Pat. No. 9,561,253, the contents ofwhich are incorporated herein by reference.

During preparation of the cellular products of the invention, cells maybe frozen at any stage. For example, cells may be frozen immediatelyafter an apheresis product is isolated from a donor but prior toseparation into fractions, after separation into fractions, aftercolumn-purification or enrichment of CD34⁺ cells, or after combiningcolumn-purified CD34⁺ cells with CD3⁺ cells.

Cryopreservation of compositions of the invention may include additionof a cryoprotectant, such as a cryoprotectant described above.Cryopreservation typically involves reducing the temperature of thecell-containing sample at a controlled rate. Cryopreservation mayinclude thawing the cell-containing sample and washing the sample toremove one or more cryoprotectants. Methods and reagents forcryopreservation, including freezing, thawing, and washing samples, areknown in the art and described in, for example, U.S. Pat. No. 9,561,253,the contents of which are incorporated herein by reference.

CD34⁺ cells may be column-purified based on qualitative or quantitativeexpression of one or more cell surface markers. Examples of suitablecell surface markers include CD34, Thy-1, CD38, and AC133. CD34⁺ cellsmay be column-purified based on the presence or absence of a marker oron the level of expression of a marker, e.g., high vs. low.

CD34⁺ cells may be column-purified by selectively binding a suitableaffinity reagent to CD34 or another marker. The affinity reagent may bean antibody, a full-length antibody, a fragment of an antibody, anaturally occurring antibody, a synthetic antibody, an engineeredantibody, a full-length affibody, a fragment of an affibody, afull-length affilin, a fragment of an affilin, a full-length anticalin,a fragment of an anticalin, a full-length avimer, a fragment of anavimer, a full-length DARPin, a fragment of a DARPin, a full-lengthfynomer, a fragment of a fynomer, a full-length kunitz domain peptide, afragment of a kunitz domain peptide, a full-length monobody, a fragmentof a monobody, a peptide, a polyaminoacid, or the like. The affinityreagent may be directly conjugated to a detection reagent and/orpurification reagent. The detection reagent and purification reagent maybe the same, or they may be different. For example, the detectionreagent and/or purification reagent may be fluorescent, magnetic, or thelike. The detection reagent and/or purification reagent may be amagnetic particle for column purification, e.g., an immunomagneticmicrosphere.

CD34⁺ cells may be column-purified from other cells using any suitablemethod. For example, CD34⁺ cells may be column-purified using animmunomagnetic column system, such as those sold under the trade nameCliniMACS by Miltenyi Biotec Inc. (Auburn, Calif.), Methods of affinitypurification of hematopoietic cells, including CD34⁺ cells, and analysisof purified populations are described in, for example, U.S. Pat. Nos.9,561,253; 9,452,184; Ng et al., Isolation of human and mousehematopoietic stem cells, Methods Mol Biol. (2009) 506:13-21. doi:10.1007/978-1-59745-409-4_2; and Spohn et al., Automated CD34⁺ cellisolation of peripheral blood stem cell apheresis product, Cytotherapy(2015) October; 17(10):1465-71. doi: 10.1016/j.jcyt.2015.04.005, thecontents of each of which are incorporated herein by reference. Themethods may include positive selection, negative selection, or both.

CD34⁺ cells may be isolated, enriched, or purified by other methods inaddition to column-purification. For example, CD34⁺ cells may beisolated, enriched, or purified by flow cytometery, cell sorting, orimmunoadsorption column separation.

CD34⁺ cells and/or CD3⁺ cells may be expanded ex vivo. Expansion mayoccur prior to, or subsequent to, freezing. Expansion may includeproviding one or more growth factors, and it may include culturing cellsin the presence of another cell type, e.g., feeder cells. Methods forexpanding hematopoietic cells are described in, for example, U.S. Pat.No. 9,561,253, the contents of which are incorporated herein byreference.

Providing Cellular Products

The cellular products of the invention may be provided to the recipientof a solid organ transplant. The cellular product may be provided by anysuitable means. For example and without limitation, the CD34⁺ cellsand/or CD3⁺ cells may be delivered to the recipient by injection using aneedle, catheter, central line or the like. In some cases, the cells maybe delivered intravascularly, intravenously, intraarterially,subcutaneously, intramuscularly, directly to the bone, or through anysource which permits the hematopoietic cells to home to an appropriatesite in the recipient such that the hematopoietic cells persist,regenerate and differentiate in the recipient. The CD34⁺ cells and/orCD3⁺ cells may be provided by infusion. The CD34⁺ cells and/or CD3⁺cells may be provided in an inpatient procedure or in an outpatientprocedure. An inpatient procedure requires admission to a hospital, andthe patient may spend one or more nights in the hospital. An outpatientprocedure does not require admission to a hospital and may be performedin a non-hospital setting, such as a clinic, doctor's office, home, orother location.

The compositions of the invention may be used in conjunction withtransplantation of any solid organ. For example and without limitation,the solid organ may be a kidney, lung, pancreas, pancreatic, isletcells, heart, intestine, colon, liver, skin, muscle, gum, eye, or tooth.The transplant may include a complete organ, a portion of an organ, orcells from a tissue of an organ. The cellular product may be providedprior to, during, or subsequent to the solid organ transplant. Forexample and without limitation, the cellular product may be providedone, two, three, four, five, or six days or one, two, three, or fourweeks prior to the solid organ transplant, or it may be provided one,two, three, four, five, or six days or one, two, three, or four weeksafter the solid organ transplant.

To facilitate establishment of mixed chimerism in the recipient, therecipient's immune system may be conditioned in conjunction withproviding the cellular product. For example, non-myeloablativeconditioning may be used. In non-myeloablative conditioning, therecipient is exposed to drugs, antibodies, irradiation, or somecombination thereof at a dose that is too low to eradicate all the bonemarrow cells. Typically, the conditioning regimen includes treatmentwith anti-thymocyte globulin (ATG), total lymphoid irradiation, andcorticosteroids (e.g. prednisone) for a period of from about 10 to 12days (e.g. for about 11 days). The irradiation may be targeted to aparticular location of the recipient's body. For example, irradiationmay be targeted to a tissue, an organ, a region of the body or the wholebody. Irradiation may be targeted to the lymph nodes, the spleen, or thethymus or any other area known to a person of skill in the art. Whenmultiple doses of irradiation are administered, the doses may betargeted to the same location or to different locations.Non-myeloablative conditioning may include the use of a T cell depletingagent, such as a monoclonal antibody or drug, e.g., fludarabine.Regimens for non-myeloablative conditioning are known in the art and aredescribed in, for example, U.S. Pat. No. 9,561,253, the contents ofwhich are incorporated herein by reference.

The methods may include immunosuppressive therapy. Immunosuppressivetherapy, or immunosuppression, involves treatment of the graft recipientwith agents that diminish the response of the host immune system againstthe donor cells, which can lead to graft rejection. Primaryimmunosuppressive agents include calcineurin inhibitors, such astacrolimus, cyclosporin A. Adjuvant agents are usually combined with acalcineurin inhibitor. Adjuvant agents include steroids, azathioprine,mycophenolic acid (MPA) agents, such as mycophenolate mofetil, mTORinhibitors, such as sirolimus, and belatacept. The use of adjuvantagents allows clinicians to achieve adequate immunosuppression whiledecreasing the dose and toxicity of individual agents. Antibody-basedtherapy may use monoclonal (e.g., muromonab-CD3) or polyclonalantibodies or anti-CD25 antibodies (e.g., basiliximab, daclizumab).Antibody-based therapy allows for avoidance or dose reduction ofcalcineurin inhibitors, possibly reducing the risk of nephrotoxicity.Regimens for immunosuppressive therapy are known in the art and aredescribed in, for example, U.S. Pat. No. 9,561,253, the contents ofwhich are incorporated herein by reference.

Immunosuppression may also diminish the response of the donor immunecells against recipient tissue, which can lead to GVHD. GVHD may beacute or chronic. Acute GVHD typically occurs in the first 3 monthsafter graft and may involve the skin, intestine, or the liver. Treatmentfor acute GVHD usually includes high-dose corticosteroids such asprednisone. Chronic GVHD typically occurs after the first 3 monthsfollowing transplant and is the major source of late treatment-relatedcomplications. Chronic GVHD may cause functional disability and requireprolonged immunosuppressive therapy.

Immunosuppressive therapy may occur in multiple phases. For example, theimmunosuppressive regimen may have an induction phase and a maintenancephase. Induction and maintenance phase strategies may use differentmedicines at doses adjusted to achieve target therapeutic levels toenhance establishment of mixed chimerism in the recipient.

Immunosuppressive therapy may be withdrawn after stable mixed chimerismhas been established in the recipient. The chimeric status of therecipient may be monitored as described below and deemed stable after acertain period, for example, 3 months, 6 months 12 months, 18 months, 24months, or longer. Thus, immunosuppression may be discontinued for therecipients after a certain period, for example, 3 months, 6 months 12months, 18 months, 24 months, or longer. Withdrawal of immunosuppressivetherapy may include tapering, i.e., progressively reducing the dosage orfrequency of treatment.

A determination of whether an individual is a full chimera, mixedchimera, or non-chimera made be made by an analysis of a hematopoieticcell sample from the solid organ transplant recipient, e.g. peripheralblood, bone marrow, etc. as known in the art. Analysis may be done byany convenient method of typing. Analysis may be performed onhematopoietic cells or a subset thereof, such as all mononuclear cells,T cells, B cells, CD56⁺NK cells, and CD15⁺ neutrophils. Chimerism can beassessed by PCR analysis of microsatellites. For example, commercialkits that distinguish polymorphisms in short terminal repeat lengths ofdonor and host origin are available. Automated readers provide thepercentage of donor type cells based on standard curves from artificialdonor and host cell mixtures.

Recipients may be categorized as fully chimeric, mixed chimeric, ornon-chimeric based on the fraction of cells that are derived from thedonor. For example, recipients can be deemed fully chimeric if they haveat least 90%, at least 95%, at least 98%, or at least 99% donor-derivedcells. Recipients can be deemed mixed chimeric if they have too fewdonor-derived cells to be categorized as fully chimeric but a fractionof donor-derived cells that exceeds a certain threshold, such as atleast 0.5%, at least 1%, at least 2%, at least 3%, at least 5%, at least7.5%, at least 10% donor-derived cells. Recipients can be deemnon-chimeric if the fraction of donor-derived cells falls below thethreshold required to be categorized as mixed chimeric.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

What is claimed is:
 1. A method of manufacturing a cellular product forestablishing mixed chimerism in a solid organ transplant recipient, themethod comprising: receiving an apheresis product containing CD34⁺ cellsand CD3⁺ cells from a solid organ transplant donor; dividing theapheresis product into a first portion and a second portion; purifyingthe first portion to obtain an enriched amount of CD34⁺ cells;introducing from the second portion to the enriched amount of CD34⁺cells (i) the obtained CD3⁺ cells in an amount greater than 1×10⁵ CD3⁺cells/kg recipient weight and ii) circulating factors from blood of thedonor, thereby creating a cellular product for establishing mixedchimerism in a solid organ transplant recipient.
 2. The method of claim1, wherein the first portion is column purified to obtain an enrichedamount of CD34⁺ cells.
 3. The method of claim 1, wherein the CD3⁺ cellsand the circulating factors introduced to the enriched amount of CD34⁺cells are not column purified from the second portion.
 4. The method ofclaim 1, wherein the cellular product comprises at least about 5×10⁵CD34⁺ cells/kg recipient weight.
 5. The method of claim 4, wherein thecellular product comprises at least about 4×10⁶ CD34⁺ cells/kg recipientweight.
 6. The method of claim 1, wherein the cellular product comprisesat least about 5×10⁵ CD3⁺ cells/kg recipient weight.
 7. The method ofclaim 6, wherein the cellular product comprises at least about 5×10⁵CD34⁺ cells/kg recipient weight.
 8. The method of claim 6, wherein thecellular product comprises at least about 4×10⁶ CD34⁺ cells/kg recipientweight.
 9. The method of claim 1, wherein the cellular product comprisesat least about 5×10⁷ CD3⁺ cells/kg recipient weight.
 10. The method ofclaim 9, wherein the cellular product comprises at least about 5×10⁵CD34⁺ cells/kg recipient weight.
 11. The method of claim 9, wherein thecellular product comprises at least about 4×10⁶ CD34⁺ cells/kg recipientweight.
 12. The method of claim 1, wherein the donor comprises a HumanLeukocyte Antigen (HLA) type that is matched to the recipient's HLAtype.
 13. The method of claim 12, wherein the donor and recipient arematched at alleles of each of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, andHLA-DR genes.
 14. The method of claim 1, wherein the donor comprises aHLA type that is mismatched to the recipient's HLA type.
 15. The methodof claim 14, wherein the donor and recipient are mismatched at at leastone allele of a gene selected from the group consisting of HLA-A, HLA-B,HLA-C, HLA-DP, HLA-DQ, and HLA-DR genes.
 16. The method of claim 1,wherein the cellular product comprises a cryoprotectant.
 17. The methodof claim 16, wherein the cryoprotectant comprises DMSO.
 18. The methodof claim 16, further comprising: cryopreserving the cellular product.19. The method of claim 1, wherein the cellular product comprises abuffer.
 20. The method of claim 19, wherein the cellular product isbuffered to a pH of from about 6.0 to about 8.0.